1. Field of the Invention
The invention relates to the fields of dermatology, cell biology, and molecular biology. More specifically, the present invention relates to drug screening assays designed to identify non-immunosuppressive agents that modulate hair growth and the use of such agents for modulation of hair growth.
2. Summary of the Related Art
The immunosuppressant drugs FK506, rapamycin and cyclosporin A are well known T-cell specific immunosuppressants that are routinely used to prevent graft rejection in organ transplant patients. In T cells, FK506 and cyclosporin A prevent calcineurin from dephosphorylating the transcription factor NF/AT (nuclear factor of activated T-cells), thereby blocking its translocation into the nucleus and preventing the receptor-mediated increase in synthesis and secretion of cytokines, such as interleukin-2 and, hence, T-cell proliferation (Heitman, J. et al., 1992, The New Biologist 4:448–460).
FK506 and cyclosporin A act by binding to endogenous intracellular receptor proteins termed immunophilins. Based on their structure and binding affinity for specific drugs, immunophilins have been divided into two classes of proteins; those proteins having an affinity for FK506 are referred to as FK506-binding proteins (FKBPs), while those having an affinity for cyclosporin are referred to as cyclophilins. Both FKBPs and cyclophilins possess a similar peptidyl-prolyl isomerase activity resulting in cis-trans-isomerization of proteins, which is believed to be important for protein folding and trafficking. In addition, both FKBPs and cyclophilins are characterized by their ability to interact with a variety of different proteins involved in signal transduction.
Several members of the FKBP family have been identified and named according to their calculated molecular mass (Lane, W. S. et al, 1991, J. Protein Chem. 10:151–160; U.S. Pat. No. 5,763,590). Cyclophilin A and FKBP12 were originally isolated as cyclosporin A and FK506 binding proteins, respectively, and were shown to exert immunosuppressive activity through inhibition of calcineurin. FKBP-51 was found to be expressed in T-cells where it inhibits calcineurin with much weaker potency, suggesting that multiple immunophilins may participate in mediating FK506 immunosuppressant activity. FKBP-51 has also been shown to be a component of the progesterone receptor complex (Nair, S. C. et al., 1997, Mol. Cell Biol. 17:594–603). FKBP52 was initially discovered as a component of the inactive steroid receptor complex (Smith, D. F. et al., 1993, J. Biol. Chem. 268:18365–71). The N-terminal domain, residues 1–149 of FKBP52, shares 55% homology with FKBP12, however, it does not have immunosuppressant activity when complexed with FK506. FKBP52 is phosphorylated by casein kinase II and has been found to have chaperone activity independent of isomerase activity (Miyata, Y. et al., 1997, Proc. Natl. Acad. Sci. USA 94:14500–14505). CyP40 has only a low affinity for cyclosporin A and is therefore capable of only slight reduction of the immunosuppressive effect of cyclosporin A.
Hsp90 is the most abundant of the heat shock proteins. A number of transcription factors and protein kinases involved in signal transduction are found complexed with hsp90 (Pratt, W. B. et al., 1999, Cell Signal 11:839–851; Pratt and Toft, 1997, Endocrine Rev. 18:306–360). When complexed with transcription factors, the hsp90 complexes are found to contain high molecular weight immunophilins with tetratricopeptide repeat (TPR) motifs (Duina, A. A. et al., 1996, Science 274:1713–1715; Bose, S. et al., 1996, Science 274:1715–1717). Such immunophilins include FKBP52 and CyP40 (Owens-Grillo, J. K., 1995, J. Biol. Chem. 270:20479–20484; Miyata, Y. et al., 1997, Proc. Natl. Acad. Sci. USA 94:14500–14505; Silverstein, A. M. et al., 1999, J. Biol. Chem. 274:36980–36986).
Causes of hair loss include aging, the action of male hormones, the loss of blood supply to hair follicles, and scalp abnormalities. In addition, genetic disposition may account for hair loss. For example, androgenic alopecia is considered to be genetically determined. Recently, a rare autosomal recessive form of hereditary alopecia, referred to as atrichia with papular lesions, was found to result from mutations in the human “hairless” gene (Ahmad, W. et al., 1998, Science 279:720–724). In individuals affected with this form of hair loss, hairs are typically absent from the scalp, and patients have very sparse eyebrows and eyelashes. Mutations in the human homolog of the mouse hairless gene lead to congenital alopecia universalis and atrichia with papular lesions. In mice bearing a mutation in the hairless gene, the hair matrix cells appear to undergo premature and massive apoptosis together with a concomitant decline in Bcl-2 expression indicating that the hairless gene product may play a role in regulating cell proliferation, differentiation and apoptosis in the hair follicle. The human hairless gene has recently been isolated and is described in WO 99/38965.
It has been reported that topical application of FK506 and cyclosporin A stimulate hair growth in a dose dependent manner (Sainsbury, T. S. L. et al., 1991, Transplant. Proc. 23:3332–3334). For example, FK506 and cyclosporin A have been shown to stimulate hair growth in experimental animals, such as mice and rats (WO 98/55090; Maurer, M., 1997, Am. J. Path. 150:1433; Yamamoto, S. et al., 1993, J. Invest. Dermatol. 102:160). The effects of FK506 and cyclosporin A and related agents have been described (Tsuji Y. et al., 1999, Exp. Dermatol. 8:366–7; McElwee, K. J. et al., 1997, Br. J. Dermatol. 137:491–7; Iwabuchi T. et al., 1995, J. Dermatol. Sci. 9:64–9; Yamamoto S. and Kato R., 1994, J. Dermatol. Sci. 7 Supp. 1:547–54; and Yamamoto S. et al., 1994, J. Invest. Dermatol 102:160–4).
In addition, Japanese patent application No. 11-174041 describes methods for identifying hair stimulating agents that can bind to immunosuppressive agent-bound proteins that can form a complex with steroid receptors, i.e., FKBP 52 or cyclophilin 40, but do not bind to FKBPs that cannot form complexes with steroid receptors, e.g., FKBP12.
The mechanism of mammalian hair growth stimulated by FK506 and cyclosporin A remains unknown. Despite their potential use as hair stimulating agents, immunosuppressive agents such as FK506 and cyclosporin A also exhibit toxic side effects such as immunosuppression. Thus, there is the need to identify and develop non-immunosuppressive agents that are useful as modulators of hair growth. The present invention is based on the discovery of the signaling pathway by which the immunosuppressive agents FK506 and cyclosporin A modulate hair growth. This discovery provides drug screening assays for identification of non-immunosuppressive agents capable of modulating hair growth.